Involvement of altered arginase activity, arginase I expression and NO production in accelerated intimal hyperplasia following cigarette smoke extract

In the present experiments, we tried to elucidate whether changes in arginase activity, protein expression of arginase-I and -II, and NO production are involved in accelerating the intimal hyperplasia following administration of cigarette smoke extract (CSE). The intimal hyperplasia was caused by removing endothelial cells with the aid of balloon embolectomy catheter in the right carotid artery of the male rabbit. The left carotid artery underwent sham operation and served as control. CSE was prepared by bubbling a stream of cigarette smoke into phosphate buffered saline. Rabbits were given subcutaneously with CSE once a day for 5 weeks from 1 week before to 4 weeks after the surgery. The specimens were assessed histologically and the intima/media ratio (%) was evaluated as an index of the intimal hyperplasia. The accelerated intimal hyperplasia with CSE was accompanied by the augmentation of the impaired cyclic GMP production, enhanced overall arginase activity and up-regulation of arginase-I. Pearson's correlation coefficient analyses revealed the close relationships among the arginase activities in endothelial cells and smooth muscle layer, the intimal/media ratio and cyclic GMP production. These results suggest that the enhanced arginase activity together with facilitated up-regulation of arginase-I with CSE, which was associated with the augmented impairment of NO production, shed a new light on the processes associated with accelerating the intimal hyperplasia in rabbit carotid arteries following CSE.     

Effect of cigarette smoke extract on arteriolar dilatation in vivo

Mayhan, William G., and Glenda M. Sharpe. Effect ofcigarette smoke extract on arteriolar dilatation in vivo.J. Appl. Physiol. 81(5):1996-2003, 1996.The goal of this study was to determine whethercigarette smoke extract alters dilatation of arterioles in vivo inresponse to agonists that produce activation of ATP-sensitive potassiumchannels and activation of adenylate cyclase. By using intravitalmicroscopy, we measured diameter of arterioles contained within themicrocirculation of the hamster cheek pouch during suffusion withagonists in the absence and presence of cigarette smoke extract (0.1, 0.5, and 1.0%). Before treatment with cigarette smoke extract,activation of ATP-sensitive potassium channels with aprikalim andcromakalim produced dose-related dilatation of cheek poucharterioles. Similarly, activation of adenylate cyclasewith isoproterenol and forskolin produced dose-related dilatation ofcheek pouch arterioles before treatment with cigarette smoke extract.Superfusion of 0.1% cigarette smoke extract did not change baselinediameter of arterioles and did not alter responses of cheek poucharterioles to activation of ATP-sensitive potassium channels andadenylate cyclase. Superfusion of 0.5 and 1.0% cigarette smoke extractalso did not alter baseline diameter of arterioles but did impairdilatation of arterioles in response to activation of ATP-sensitivepotassium channels and adenylate cyclase. These findings suggest thatcigarette smoke extract impairs dilatation of resistance arterioles inresponse to activation of important cellular dilator pathways.

     

Up-regulation of protein tyrosine nitration in methamphetamine-induced neurotoxicity through DDAH/ADMA/NOS pathway

Protein tyrosine nitration is an important post-translational modification mediated by nitric oxide (NO) associated oxidative stress, occurring in a variety of neurodegenerative diseases. In our previous study, an elevated level of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein was observed in different brain regions of acute methamphetamine (METH) treated rats, indicating the possibility of an enhanced expression of protein nitration that is mediated by excess NO through the DDAH1/ADMA (Asymmetric Dimethylated l-arginine)/NOS (Nitric Oxide Synthase) pathway. In the present study, proteomic methods, including stable isotope labeling with amino acids in cell culture (SILAC) and two dimensional electrophoresis, were used to determine the relationship between protein nitration and METH induced neurotoxicity in acute METH treated rats and PC12 cells. We found that acute METH administration evokes a positive activation of DDAH1/ADMA/NOS pathway and results in an over-production of NO in different brain regions of rat and PC12 cells, whereas the whole signaling could be repressed by DDAH1 inhibitor N^ω-(2-methoxyethyl)-arginine (l-257). In addition, enhanced expressions of 3 nitroproteins were identified in rat striatum and increased levels of 27 nitroproteins were observed in PC12 cells. These nitrated proteins are key factors for Cdk5 activation, cytoskeletal structure, ribosomes function, etc. l-257 also displayed significant protective effects against METH-induced protein nitration, apoptosis and cell death. The overall results illustrate that protein nitration plays a significant role in the acute METH induced neurotoxicity via the activation of DDAH1/ADMA/NOS pathway.     

Bone marrow mononuclear cells up-regulate toll-like receptor expression and produce inflammatory mediators in response to cigarette smoke extract

Several reports link cigarette smoking with leukemia. However, the effects of cigarette smoke extract (CSE) on bone marrow hematopoiesis remain unknown. The objective of this study was to elucidate the direct effects of cigarette smoke on human bone marrow hematopoiesis and characterize the inflammatory process known to result from cigarette smoking. Bone marrow mononuclear cells (BMCs) from healthy individuals when exposed to CSE had significantly diminished CFU-E, BFU-E and CFU-GM. We found increased nuclear translocation of the NF-κB p65 subunit and, independently, enhanced activation of AKT and ERK1/2. Exposure of BMCs to CSE induced IL-8 and TGF-β1 production, which was dependent on NF-κB and ERK1/2, but not on AKT. CSE treatment had no effect on the release of TNF-α, IL-10, or VEGF. Finally, CSE also had a significant induction of TLR2, TLR3 and TLR4, out of which, the up-regulation of TLR2 and TLR3 was found to be dependent on ERK1/2 and NF-κB activation, but not AKT. These results indicate that CSE profoundly inhibits the growth of erythroid and granulocyte-macrophage progenitors in the bone marrow. Further, CSE modulates NF-κB- and ERK1/2-dependent responses, suggesting that cigarette smoking may impair bone marrow hematopoiesis in vivo as well as induce inflammation, two processes that proceed malignant transformation.     

Changes in rabbit thymus lymphoid tissue after administration of pyrogenal and hydrocortisone]

After a single administration of pyrogenal (0.2 and 5 mkg/kg) and hydrocortisone (100 mg/kg) histological changes in the rabbit thymus have been studied and compared with the level of 11-oxycorticosteroids (11-OCS) in blood of the animals under investigation. Hydrocortisone, while producing a considerable rise in the level of 11-OCS, increases the number of degenerating cells, inhibits mitotic activity and decreases the amount of lymphocytes per stipulated area unit in the cortical substance. Changes produced in the thymus after pyrogenal administration are similar to those produced by hydrocortisone administration but are pronounce much weaker, that is connected with concentration of corticosteroids circulating in blood.     

Inhibition of chloride secretion in human bronchial epithelial cells by cigarette smoke extract

Chronic bronchitis, a disease mainly of cigarette smokers, shares many clinical features with cystic fibrosis, a disease of altered ion transport, suggesting that the negative effects of cigarette smoke on mucociliary clearance may be mediated through alterations in ion transport. We tested the hypothesis that cigarette smoke extract would inhibit chloride secretion in human bronchial epithelial cells. In agreement with studies in canine trachea, cigarette smoke extract inhibited net chloride secretion without affecting sodium transport. We performed microelectrode impalements and impedance analysis studies to investigate the physiological mechanisms of this inhibition. These data demonstrated that cigarette smoke extract caused an acute increase in membrane resistances in conjunction with apical membrane hyperpolarization, an effect consistent with inhibition of an apical membrane anion conductance. After this acute phase, both membrane resistances decreased while membrane potentials continued to hyperpolarize, indicating that cigarette smoke extract also inhibited the basolateral entry of chloride into the cell. Furthermore, cigarette smoke extract caused an increase in mucin secretion. Therefore, the ion transport phenotype of human bronchial epithelial cells exposed to cigarette smoke extract is similar to that of cystic fibrosis epithelia in which there is sodium absorption out of proportion to chloride secretion in the setting of increased mucus secretion.     

Alterations in myocardial ultrastructure and protein expression after a single injection of isoproterenol

Immunochemical and electron microscopic characterization of rat myocardium was conducted 2 h and 3 weeks after a single injection of isoproterenol in rats. The relative content of several myospecific proteins (KRP – kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, myosin light chain kinase – MLCK) and extracellular matrix protein fibronectin was determined by immunoblotting. Two hours after injection of 50 mg/kg isoproterenol a destruction of some cardiomyocytes, contracture of myofibrils and mild edema of intercellular space was observed. The content of all the studied proteins except KRP decreased below control levels. This situation sustained 3 weeks after injection and paralleled alterations in cardiomyocyte ultrastructure. Areas of myofibrillar contracture and lysis were noted, glycogen granules were sparse; mitochondria contained arrow-like inclusions that are characteristic for calcium overload, also huge mitochondria contacting each other by specialized intermitochondrial contacts were detected. Clumps of unripe elastic fibers in enlarged intercellular space were combined with increased deposition of collagens type I and III forming areas of fibrosis. The smaller dosage of isoproterenol (10 mg/kg) rendered no significant damage in the acute postinjection period but 3 weeks later it induced the thickening of extracellular matrix around cardiac cells and the increase in KRP and tubulin content by 26 and 32%, correspondingly. MLCK levels remained depressed throughout the experiment. The rise in KRP expression was also observed after the addition of isoproterenol to cultured chicken embryo cardiomyocytes. Obtained results indicate that even a single injection of isoproterenol creates long lasting structural alterations in cardiac muscle accompanied by the increased expression of extracellular matrix proteins and several sarcoplasmic proteins apparently involved in hypertrophic response of cardiomyocytes.     

Alterations in gene expressions encoding preproET-1 and NOS in pulmonary tissue in endotoxemic rats

Septic shock is characterized by hypotension and a hyporeactive response to vasopressor agents. The pathogenesis is due to vascular leaks and an increased synthesis of cytokines and nitric oxide (NO). The present study examined the time-dependent alterations of endothelin-1 (ET-1) and the expression of NO synthase (NOS) in lung tissue in a septic rat model. Normal Sprague-Dawley (SD) rats aged 10 weeks received 15 mg/kg lipopolysaccharide (LPS) and then were sacrificed at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. Both systolic and diastolic pressure decreased in SD rats after LPS administration. Time-dependent onset of features of acute lung injury, such as the infiltration of inflammatory cells and thickening of alveolar septa, were seen in rats that received LPS. A 2.8-fold increase in the expression of preproET-1 level was observed in lung tissue 6 hrs after LPS administration. The expression of endothelial NOS (eNOS) was also altered in lung tissue in a time-dependent fashion. After the administration of LPS, there was a 16-fold increase in the expression of eNOS mRNA. The peak expression of inducible NOS (iNOS) in lung tissue specimens obtained from rats that received LPS was 45-fold higher than that in control rats. ET-1 is a potent vasoconstrictor and thereby may play an important role in the pathogenesis of acute lung injury in a septic rat model. The increased expression of NOS may result in excess NO production and may also play a role in the pulmonary complications of endotoxemia.     

Phospholipid fatty acid remodeling and carbonylated protein increase in extracellular vesicles released by airway epithelial cells exposed to cigarette smoke extract

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.     

Alterations in regional ventilation, perfusion, and shunt after smoke inhalation measured by PET.

Regional changes in ventilation and perfusion occurring in the early hours after smoke inhalation injury were evaluated through the use of positron emission tomography. Five lambs were imaged before and 1, 2, and 4 h after receiving 100 breaths of cotton smoke. Utilizing a recently developed model of (13)N tracer kinetics (3), we evaluated changes in ventilation, perfusion, shunt, and regional gas content in nondependent, middle, and dependent lung zones. The data demonstrated a progressive development of regional shunt in dependent (dorsal) regions in which perfusion remained the highest throughout the study. These findings, together with decreasing regional ventilation and fractional gas content in the dependent regions, correlated with decreasing arterial Pa(O(2)) values over the course of the study. A negative correlation between regional shunt fraction and regional gas content in dependent and middle regions suggests that shunt was caused by progressive alveolar derecruitment or flooding.     

Epithelial expression of TLR4 is modulated in COPD and by steroids,salmeterol and cigarette smoke

The toll-like receptors (TLRs) are a key component of host defense in the respiratory epithelium. Cigarette smoking is associated with increased susceptibility to infection, while COPD is characterised by bacterial colonisation and infective exacerbations. We found reduced TLR4 gene expression in the nasal epithelium of smokers compared with non-smoking controls, while TLR2 expression was unchanged. Severe COPD was associated with reduced TLR4 expression compared to less severe disease, with good correlation between nasal and tracheal expression. We went on to examine the effect of potential modulators of TLR4 expression in respiratory epithelium pertinent to airways disease. Using an airway epithelial cell line, we found a dose-dependent downregulation in TLR4 mRNA and protein expression by stimulation with cigarette smoke extracts. Treatment with the corticosteroids fluticasone and dexamethasone resulted in a dose-dependent reduction in TLR4 mRNA and protein. The functional significance of this effect was demonstrated by impaired IL-8 and HBD2 induction in response to LPS. Stimulation with salmeterol (10^-6 M) caused upregulation of TLR4 membrane protein presentation with no upregulation of mRNA, suggesting a post-translational effect. The effect of dexamethasone and salmeterol in combination was additive, with downregulation of TLR4 gene expression, and no change in membrane receptor expression. Modulation of TLR4 in respiratory epithelium may have important implications for airway inflammation and infection in response to inhaled pathogens.     

Effect of litter size and bacitracin administration on tissue protein synthesis of lactating rabbit does

Bacitracin is an antibiotic used in rabbit husbandry to control microbial digestive pathologies. Collateral effects on absorption and mucosal development have been reported and these may impact on protein metabolism. This study aims to analyse the effect of the antibiotic on protein synthesis in lactating does because mammary gland metabolism and milk output should provide a sensitive index of any undesirable action of bacitracin. Rates of protein synthesis were measured in mammary gland, liver, intestinal mucosa and muscle of lactating rabbits does by injecting a flooding dose of [(2)H(5)]phenylalanine into the auricular artery of two groups (each n = 8) of New Zealand White does fed different experimental diets. The control group (C) received the basal diet and the bacitracin group (B) ingested the same diet but supplemented with bacitracin (100 mg/kg). Animals received the experimental diet from day 28 of pregnancy until day 26 of lactation when they were slaughtered. Just after birth, litter size was adjusted by cross-fostering either to five or nine pups (four does per dietary treatment). The relative weight of the liver tended to be greater in those females receiving the B diet (27 v. 22.5 g/kg BW; P < 0.07), while diet did not affect mammary gland weight (255.7 ± 10.59 g). Fractional protein synthesis rate (FSR) was higher for intestinal mucosa (duodenum; 51.7% ± 2.09%/day) followed by mammary gland and liver (38.29 ± 2.62%/day and 40.2 ± 1.98%/day, respectively), and the lowest value was observed in muscle (2.92 ± 0.26%/day; P < 0.0001). Bacitracin treatment lowered FSR in the mammary gland by 23% (P = 0.024) and this was independent of litter size. Conversely, FSR in the duodenum was not affected by antibiotic treatment but reduced by 15% (P = 0.021) for the larger litter size.     

Dose-responsiveness and persistence of microRNA expression alterations induced by cigarette smoke in mouse lung

Our previous studies demonstrated that exposure to cigarette smoke (CS), either mainstream or environmental, results in a remarkable downregulation of microRNA expression in the lung of both mice and rats. The goals of the present study were to evaluate the dose responsiveness to CS and the persistence of microRNA alterations after smoking cessation. ICR (CD-1) neonatal mice were exposed whole-body to mainstream CS, at the doses of 119, 292, 438, and 631mg/m(3) of total particulate matter. Exposure started within 12h after birth and continued daily for 4 weeks. The levels of bulky DNA adducts and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were measured by (32)P postlabeling procedures, and the expression of 697 mouse microRNAs was analyzed by microarray. The highest CS dose was lethal. Exposure to CS caused a dose-dependent increase of DNA alterations. DNA adducts and, even more sharply, 8-oxodGuo were reverted 1 and 4 weeks after smoking cessation. Exposure to CS resulted in an evident dysregulation of microRNA expression profiles, mainly in the sense of downregulation. The two lowest doses were not particularly effective, while the highest nonlethal dose produced extensive microRNA alterations. The expression of most downregulated microRNAs, including among others 7 members of the let-7 family, was restored one week after smoking cessation. However, the recovery was incomplete for a limited array of microRNAs, including mir-34b, mir-345, mir-421, mir-450b, mir-466, and mir-469. Thus, it appears that microRNAs mainly behave as biomarkers of effect and that exposure to high-dose, lasting for an adequate period of time, is needed to trigger the CS-related carcinogenesis process in the experimental animal model used.     

Chromosomal localization, gene structure, and expression pattern of DDAH1: comparison with DDAH2 and implications for evolutionary origins

Endogenously produced asymmetrically methylated arginine residues are competitive inhibitors of all three isoforms of nitric oxide synthase (NOS). The enzyme dimethylarginine dimethylaminohydrolase (DDAH) specifically hydrolyzes these asymmetrically methylated arginine residues to citrulline and methylamines. Previously we have proposed that regulation of asymmetric methylarginine concentration by DDAH may provide a novel mechanism for the regulation of NOS activity in vivo. Recently we reported the cloning of human DDAH and identified a novel human DDAH isoform (DDAH I and DDAH II, respectively). Here we report that the DDAH1 gene maps to chromosome 1p22 and confirm that DDAH2 maps to the MHC III region of chromosome 6p21.3. Extensive analysis of the distribution of DDAH1 and DDAH2 mRNA in 50 human tissues indicates differential expression of DDAH isoforms in brain regions, in immune cells, and during development. DDAH2 expression predominates in highly vascularized tissues that express the endothelial NOS isoform and in immune tissues that can express iNOS. Whereas DDAH2 is expressed at relatively high levels in all fetal tissues examined, DDAH1 expression varies little between fetal and adult tissues. The chromosomal localization of the DDAHs is consistent with gene duplication, and consistent with this, comparison of the gene structures indicates that the intron/exon organization is highly conserved. Phylogenetic analysis of DDAH sequences from diverse species suggests that DDAH gene duplication occurred prior to the emergence of bony fish some 400 million years ago. Overall the data suggest that DDAH2 may be the more ancient of the two genes.     

Aromatase inhibitors in cigarette smoke, tobacco leaves and other plants

A chance observation that cigarette smoke interferes with the aromatase assay led us to investigate tobacco leaf and smoke extracts for the presence of aromatase inhibitors. The highest inhibitory activity was found in the basic fraction of cigarette smoke. Further purification of this fraction led to the identification of N-n-octanoylnornicotine. Synthesis and testing of a series of acylated nornicotines and anabasines for their ability to inhibit aromatase showed an interesting correlation of activity with the length of the acyl carbon chain, with maximum activity at C-11. The acylated derivatives showed activity which was significantly greater than that of nicotine and anabasine. In vivo studies in rats indicated that administration of this inhibitor delayed the onset of NMU-induced breast carcinoma and altered the estrus cycle. These in vivo studies suggest that tobacco alkaloid derivatives exert their effects by suppression of the aromatase enzyme system. Toxicity studies indicated relatively low toxicity with LD50 for N-n-octanoylnornicotine = 367 mg/kg body weight. When extracts from thirty five varieties of vegetables, plant leaves, and fruits were analyzed, seventeen showed quantitatively significant aromatase inhibition which was comparable to that of green tobacco leaf, suggesting that naturally occurring substances may affect endocrine function through aromatase inhibition.     

Respiratory effects of cigarette smoke, dust, and histamine in newborn rabbits

We studied the respiratory effects of cigarette smoke, 5% histamine aerosol, and dust in unanesthetized 1- to 7-day-old rabbits in a body plethysmograph. Cigarette smoke immediately provoked the animal's arousal and irregular breathing. Histamine and dust had no effect in some of the youngest animals. In others, 5-15 s from the onset of the exposure to either of the two stimuli, respiratory rate increased and the depth of breathing decreased. These changes were more pronounced with age. The fact that effects of dust and aerosol lessened with time of exposure showed adaptation to the stimuli. The age dependence of the reflex response was also observed after injection of 50 micrograms of histamine per kilogram into the external jugular vein in anesthetized (50 mg ketamine + 3 mg acepromazine per kg) and tracheostomized rabbits during the 1st wk of life. In 1-day-old animals, a short-lasting excitation was followed by apnea or a prolongation of expiratory phase. Peak amplitude of the diaphragmatic EMG (EMGdi) increased in all animals, but only in the youngest was the EMGdi increase paralleled by an increase in tidal volume. In vagotomized animals or animals pretreated with H1-blocker, histamine never affected timing parameters in animals greater than 1 day old. In the youngest animals, respiratory depression due to histamine was not abolished after vagotomy or promethazine. The results imply that inputs from the upper airways and the rapidly adapting pulmonary mechanoreceptors exert their effects on the pattern of breathing immediately after birth in rabbits. The importance of those inputs increases with maturation.     

Inhibition of lecithin:Cholesterol acyltransferase activity in human blood plasma by cigarette smoke extract and reactive aldehydes

Cigarette smoking is a risk factor for atherosclerosis. It is conceivable that reactive chemical components in cigarette smoke may adversely affect reverse cholesterol transport at the level of lecithin:cholesterol acyltransferase (LCAT) and promote atherogenesis. Hence, the effect of cigarette smoke extract (CSE) on the activity of LCAT in human plasma was studied. When incubated with plasma, CSE caused both concentration- and time-dependent losses of LCAT activity. Addition of glutathione, but not ascorbate, to plasma prevented loss of LCAT activity caused by CSE. Incubation of plasma with some reactive aldehydes known to be present in cigarette smoke also inhibited LCAT activity. Among five aldehydes tested, acrolein was the strongest inhibitor of LCAT, with complete enzyme inhibition occurring at 1 mM. Acetaldehyde was the weakest inhibitor of LCAT, with 85% enzyme inhibition at 50 mM. Hexanal, formaldehyde, and malondialdehyde completely inhibited LCAT activity at 10, 50, and 50 mM, respectively. When plasma was incubated with 1 mM acrolein in the presence of 2.5 mM glutathione or dihydrolipoic acid, 100 and 57% of LCAT activity, respectively, remained after incubation. This finding suggests that reactive aldehydes may form adducts with certain free sulfhydryl groups functioning in the active site of LCAT to inhibit enzyme activity. It is concluded that reactive aldehydes are at least partially responsible for the reduction in LCAT activity in plasma treated with CSE.